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P Barbosa

Publications and source records attributed to P Barbosa.

At least 19 recordsLinked to original sources
PubMed1998

Recognition of two overlapping CTL epitopes in HIV-1 p17 by CTL from a long-term nonprogressing HIV-1-infected individual.

HIV-1 infection has been shown to elicit strong CTL responses in some infected persons, but few data are available regarding the relationship between targeted epitopes and in vivo viral quasispecies. In this study, we examined the CTL response in a person infected for 15 yr with a CD4 count persistently >500 cells/microl. The dominant in vivo activated CTL response was directed against two overlapping Gag CTL epitopes in an area of p17 known to be essential for viral replication. The 9-mer SLYNTVATL (amino acids 77-85) was recognized in conjunction with HLA-A2, whereas the overlapping 8-mer TLYCVHQR (amino acids 83-91) was recognized by HLA-A11-restricted CTL. Analysis of in vivo virus sequences both in PBMC and plasma revealed the existence of sequence variation in this region, which did not affect viral replication in vitro, but decreased recognition by the A11-restricted CTL response, with maintenance of the A2-restricted response. These results indicate that an essential region of the p17 protein can be simultaneously targeted by CTL through two different HLA molecules, and that immune escape from CTL recognition can occur without impairing viral replication. In addition, they demonstrate that Ag processing can allow for presentation of overlapping epitopes in the same infected cell, which can be affected quite differently by sequence variation.

Amino Acid Sequence↗
PubMed1998

Characterization of plantar verrucae among individuals with human immunodeficiency virus.

Plantar verrucae, caused by human papillomavirus (HPV), are commonly found in patients who have tested positive for the antibodies to human immunodeficiency virus (HIV). A better understanding of the characteristics of plantar verrucae in HIV+ patients in needed. A pilot study was conducted concentrating on three characteristics--the size, the number, and the clinical type--of verrucae present in this population. These parameters were studied in HIV+ and HIV- populations, and they were evaluated in relation to the CD4 levels of HIV+ individuals. The HIV+ individuals presented with plantar verrucae that were larger and more numerous than those found in HIV- individuals. The HIV+ population presented with all three clinical types of plantar verrucae and had significantly more mosaic-type warts than did HIV- individuals. The three characteristics did not correlate with CD4 cell counts, suggesting that the severity and extent of HPV infection do not depend on the level of immunosuppression of the HIV+ patient.

Adult↗
PubMed1998

[Uveitis].

Uveitis is a general term that refers to the inflammation of uveal tract, which is an important cause of blindness in young people. It is well known that uveitis can be the initial manifestation of a systemic disease (S.D.), and may appear years before the diagnosis of the primary disease. Uveitis should be integrated in a systemic study with proper testing. Therefore, the diagnosis is a matter for the ophthalmologist and the Specialist in internal medicine. We have made a retrospective study of 71 patients with chronic uveitis or panuveitis. We found 54.9% of primary uveitis and 45.1% of S.D. associated uveitis, most of them with Behçet's disease (16/71) and Ankylosing Spondilytis (7/71). HLA typing of the patients showed a decreased frequency of HLA A1 and HLA A3 antigens and an increased frequency of the HLA B27 antigen, when compared to a Portuguese control population. We confirmed the important role of HLA B27 as an independent susceptibility factor for anterior uveitis. The lowest HLA A3 frequency was observed in the group of S.D. associated uveitis, which could suggest that this antigen may play a role as a factor of resistance to uveitis.

Adolescent↗
PubMed1998

Molecular biology of HIV.

This article describes the molecular aspects of the life cycle of HIV. Evolution theories of HIV origin, specific steps in the infection cycle, and regulation of expression are discussed. The article concludes with a summary of the current theories of the pathogenesis of HIV infection and the breakdown of the immune system.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Plantar verrucae and HIV infection.

No single treatment of plantar verrucae has proven to be effective in all situations, which represents a greater challenge when treating patients infected with HIV. In this patient population, plantar verrucae appear to be more aggressive, and recurrence is frequently observed. Mosaic-type plantar verrucae, which are usually more resistant to treatment, are common in this population. Factors including the patient's general health, number of warts, location of warts, effect of lesions on daily activities, and sensitivity should be carefully analyzed before deciding on a treatment option. Among the many treatment modalities available for plantar verrucae, three frequently are used by podiatrists for patients infected with HIV: Histofreezer cryotherapy, bleomycin sulfate intralesional injections, and surgical intervention by curettage. These three modalities are recommended; however, all approaches described in this article should be kept as viable options. The patient population infected with HIV represents a unique group of patients, and choices in treatments for these patients should be made by taking into account all ramifications of HIV infection.

Bleomycin↗
PubMed1997

The nef gene products of both simian and human immunodeficiency viruses enhance virus infectivity and are functionally interchangeable.

Adult rhesus macaques infected with nef-defective simian immunodeficiency virus (SIV) exhibit extremely low levels of steady-state virus replication, do not succumb to immunodeficiency disease, and are protected from experimental challenge with pathogenic isolates of SIV. Similarly, rare humans found to be infected with nef-defective human immunodeficiency virus type 1 (HIV-1) variants display exceptionally low viral burdens and do not show evidence of disease progression after many years of infection. HIV-1 Nef induces the rapid endocytosis and lysosomal degradation of cell surface CD4 and enhances virus infectivity in primary human T cells and macrophages. Although expression of SIV Nef also leads to down-modulation of cell surface CD4 levels, no evidence for SIV Nef-induced enhancement of virus infectivity was observed in earlier studies. Thus, it remains unclear whether fundamental differences exist between the activities of HIV-1 and SIV Nef. To establish more clearly whether the SIV and HIV-1 nef gene products are functionally analogous, we compared the replication kinetics and infectivity of variants of SIVmac239 that either do (SIVnef+) or do not (SIV delta nef) encode intact nef gene products. SIVnef+ replicates more rapidly than nef-defective viruses in both human and rhesus peripheral blood mononuclear cells (PBMCs). As previously described for HIV-1 Nef, SIV Nef also enhances virus infectivity within each cycle of virus replication. As a strategy for evaluating the in vivo contribution of HIV-1 nef alleles and long terminal repeat regulatory sequences to the pathogenesis of immunodeficiency disease, we constructed SIV-HIV chimeras in which the nef coding and U3 regulatory regions of SIVmac239 were replaced by the corresponding regions from HIV-1/R73 (SIVR7nef+). SIVR7nef+ displays enhanced infectivity and accelerated replication kinetics in primary human and rhesus PBMC infections compared to its nef-defective counterpart. Converse chimeras, containing SIV Nef in an HIV-1 background (R7SIVnef+) also exhibit greater infectivity than matched nef-defective viruses (R7SIV delta nef). These data indicate that SIV Nef, like that of HIV-1, does enhance virus replication in primary cells in tissue culture and that HIV-1 and SIV Nef are functionally interchangeable in the context of both HIV-1 and SIV.

Animals↗
PubMed1996

Cytotoxic T lymphocytes in asymptomatic long-term nonprogressing HIV-1 infection. Breadth and specificity of the response and relation to in vivo viral quasispecies in a person with prolonged infection and low viral load.

Although vigorous activated and memory CTL have been associated with HIV-1 infection, data are lacking regarding the breadth of epitopes recognized in a given individual and the relationship to the viral quasispecies present in vivo. In this study we performed a detailed analysis of the HIV-1-specific CTL response in a seropositive person with documented HIV-1 infection of 15 yr duration, stable CD4 counts above 500 cells/ml, and viral load persistently below 500 molecules of RNA/ml of plasma. Epitope mapping studies revealed the presence of HLA class I-restricted CTL responses to six different epitopes in p17, p24, RT, Env, and Nef, which conferred broadly cross-reactive recognition of reported HIV-1 variants. Sequence analysis of autologous viruses revealed the absence of immune escape variants within five of the six epitopes. Despite consistently low viral RNA levels in plasma and viral DNA levels in PBMC, in vivo-activated circulating CTL were detected against three of the epitopes. Five of the six epitopes, including the three dominant epitopes, have been detected in persons with progressive disease, suggesting that nonprogressors may not target unique epitopes. This study demonstrates that HIV-1-specific CTL can be highly activated and broadly directed in the setting of an extremely low viral load, and that neither high viral load nor antigenic diversity is required for the generation of a multispecific CTL response. Although the detection of strong CTL responses, low viral load, and lack of immune escape are consistent with the hypothesis that CTL may contribute to lack of disease progression in this individual, the contribution of these responses to maintenance of the asymptomatic state remains to be determined.

Amino Acid Sequence↗
PubMed1996

Recognition of the highly conserved YMDD region in the human immunodeficiency virus type 1 reverse transcriptase by HLA-A2-restricted cytotoxic T lymphocytes from an asymptomatic long-term nonprogressor.

The human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) is an important target for therapeutic intervention and for HIV-1-specific cytotoxic T lymphocytes (CTL). An HLA-A2-restricted CTL epitope containing the sequence YMDD, which is highly conserved among human and animal retroviruses and essential for function of the RNA-dependent DNA polymerase, is identified. The drug resistance mutation at RT amino acid 184 (M184V), associated with (-)-2'-deoxy-3'-thiacytidine (lamivudine), (-)-2'-deoxy-5-fluoro-3'-thiacytidine (FTC), and dideoxyinosine resistance, is located within this epitope and abolishes recognition by an established CTL response. This study demonstrates that the CTL response may target functionally relevant regions of the RT protein and suggests drug therapy may select for viral variants with altered susceptibility to established cellular immune responses.

Amino Acid Sequence↗
PubMed1995

Activation of virus replication after vaccination of HIV-1-infected individuals.

Little is known about the factors that govern the level of HIV-1 replication in infected individuals. Recent studies (using potent antiviral drugs) of the kinetics of HIV-1 replication in vivo have demonstrated that steady-state levels of viremia are sustained by continuous rounds of de novo infection and the associated rapid turnover of CD4+ T lymphocytes. However, no information is available concerning the biologic variables that determine the size of the pool of T cells that are susceptible to virus infection or the amount of virus produced from infected cells. Furthermore, it is not known whether all CD4+ T lymphocytes are equally susceptible to HIV-1 infection at a given time or whether the infection is focused on cells of a particular state of activation or antigenic specificity. Although HIV-1 replication in culture is known to be greatly facilitated by T cell activation, the ability of specific antigenic stimulation to augment HIV-1 replication in vivo has not been studied. We sought to determine whether vaccination of HIV-1-infected adults leads to activation of virus replication and the targeting of vaccine antigen-responsive T cells for virus infection and destruction. Should T cell activation resulting from exposure to environmental antigens prove to be an important determinant of the steady-state levels of HIV-1 replication in vivo and lead to the preferential loss of specific populations of CD4+ T lymphocytes, it would have significant implications for our understanding of and therapeutic strategies for HIV-1 disease. To begin to address these issues, HIV-1-infected individuals and uninfected controls were studied by measurement of immune responses to influenza antigens and quantitation of virion-associated plasma HIV-1 RNA levels at baseline and at intervals after immunization with the trivalent influenza vaccine. Influenza vaccination resulted in readily demonstrable but transient increases in plasma HIV-1 RNA levels, indicative of activation of viral replication, in HIV-1-infected individuals with preserved ability to immunologically respond to vaccine antigens. Activation of HIV-1 replication by vaccination was more often seen and of greater magnitude in individuals who displayed a T cell proliferative response to vaccine antigens at baseline and in those who mounted a significant serologic response after vaccination. The fold increase in viremia, as well as the rates of increase of HIV-1 in plasma after vaccination and rates of viral decline after peak viremia, were higher in individuals with higher CD4+ T cell counts.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1995

Exploring the role of histidines in the catalytic activity of duck delta-crystallins using site-directed mutagenesis.

The duck delta 2-crystallin gene encodes an enzymatically-active argininosuccinate lyase while the delta 1-crystallin gene product, although 94% identical, is enzymatically inactive. Four histidine residues in the duck delta 2-crystallin. His91, His110, His162 and His178, were converted to asparagine residues in an effort to define the role of histidines in the catalytic process of this enzyme-crystallin and to explain the lack of enzyme activity in the delta 1-crystallin protein. The recombinant mutant proteins were expressed in E. coli and purified to homogeneity for analysis. These four residues were chosen because they fall within highly conserved regions of argininosuccinate lyases from several species. This analysis revealed that change of His91 or His162 for asparagine resulted in complete loss of activity. The His110 enzyme had a reduced Vmax and the His178 enzyme was near normal in its kinetic properties. These data confirm the roles of histidine in the catalytic process of this enzyme-crystallin and suggest that the change of His91 to Gln91 observed in the duck and chicken delta 1-crystallin molecules may be sufficient to account for the lack of enzymatic activity of those proteins.

Amino Acid Sequence↗
PubMed1994

Kinetic analysis of HIV-1 early replicative steps in a coculture system.

During an acute human immunodeficiency virus (HIV) infection in vitro, different forms of unintegrated viral DNA accumulate in target cells. They include linear full-length HIV DNA molecules, which are the precursors of the integrated provirus, and two types of circular molecules (with one or two LTRs), whose role and mode of formation are not fully understood. To evaluate the intracellular fate of HIV unintegrated DNA, and to follow the formation of the two types of circular DNA molecules, the nuclear transport of viral DNA, and its integration in host cell DNA, we have designed a "DNA chase" assay. This assay is based on cocultivation of persistently HIV-1-infected H9 cells with uninfected MT4, allowing rapid accumulation of viral DNA, which is then blocked by addition of AZT. In this highly efficient, synchronous, one-step cycle infection system, HIV linear DNA can be detected on Southern blots as early as 4 hr after the start of the coculture. Subsequently, viral DNA that had been synthesized before the addition of AZT could be "chased," establishing that almost all linear DNA molecules are rapidly transported to the nucleus, where they are either processed into the two types of circles or integrated. We could estimate that from the number of viral DNA molecules synthesized in 6 hr in this system, at least a third will become integrated and another third will circularize within 24 hr.

Biological Transport↗
PubMed1993

[Mitral valvuloplasty with Inoue balloon catheter. Analysis of 112 cases].

PURPOSE: To present immediate results of percutaneous mitral valvuloplasty (PVM) with Inoue balloon for the treatment of mitral stenosis. METHODS: One hundred and twelve consecutive patients mean age 31.26 +/- 9.5 years, 82% females, the majority in functional class III/IV (69%) underwent percutaneous mitral valvuloplasty (PVM) with Inoue balloon. The procedure was undertaken by the transseptal technique. RESULTS: PMV achieved significant reduction in mean left atrial pressure pre 24.57 +/- 5.37 post 12.36 +/- 4.82, mitral gradient pre 15.31 +/- 4.28 post 3.48 +/- 2.09 and increased mitral valve area pre 0.88 +/- 0.12 post 2.0 +/- 0.28 (P < 0.001). An increase of at least 25% of the mitral valve area with a final result greater than 1.5 cm2 was obtained in 96% of the cases. Cardiac tamponade occurred in five cases (4.4%). In 4 a successful treatment was accomplished in the catheterization laboratory. Significant mitral regurgitation was seen in one case (0.9%) and there was one death (0.9%). CONCLUSION: The results of this series attest to the effectiveness of the PMV with Inoue balloon in selected cases of mitral stenosis.

Adolescent↗
PubMed1991

Expression of duck lens delta-crystallin cDNAs in yeast and bacterial hosts. Delta 2-crystallin is an active argininosuccinate lyase.

The major soluble protein in the lenses of most birds and reptiles is delta-crystallin. In chickens and ducks the delta-crystallin gene has duplicated, and in the duck both genes contribute to the protein in the lens, while in the chicken lens there is a great preponderance of the delta 1 gene product. Purified delta-crystallin has previously been shown to possess the enzymatic activity of argininosuccinate lyase. In order to determine the enzymatic properties of the two duck delta-crystallins their corresponding cDNA molecules were placed in yeast and bacterial expression plasmids. In Saccharomyces cerevisiae, the activity of each crystallin was assessed by transformation of the expression plasmids into a strain deficient for argininosuccinate lyase activity. The ability of the resulting yeast to grow on arginine deficient medium was used as a measure of enzymatic activity. Yeast expressing the duck delta 2-crystallin protein grew rapidly, while those expressing delta 1-crystallin failed to grow. Enzyme activity measurements confirmed the presence of activity in the delta 2-crystallin-expressing yeast, and no detectable activity could be demonstrated in the delta 1-crystallin-expressing yeast. Northern blotting of RNA from the transformed yeast revealed equal levels of mRNA species from the two constructs. For further analysis, the delta 2-crystallin cDNA was placed in the bacterial expression plasmid, pET-3d. The delta 2-crystallin protein produced in Escherichia coli was purified to homogeneity and analyzed to determine the kinetic properties. A Km of 0.35 mM was determined for argininosuccinate and a Vm of 3.5 mumols/min/mg was determined. These data demonstrate that, following duplication of the primordial argininosuccinate lyase gene, one of the genes maintained its role as an enzyme (delta 2-crystallin) while also serving as a crystallin and the other has evolved to specialize as a structural protein in the lens (delta 1-crystallin), presumably losing most or all of its catalytic capacity.

Animals↗
PubMed1991

Analysis of naturally occurring and site-directed mutations in the argininosuccinate lyase gene.

Argininosuccinic aciduria is an inborn error of metabolism due to the genetic deficiency of argininosuccinate lyase. In order to determine the molecular basis for the disease, RNA isolated from cultured skin fibroblasts derived from four unrelated patients was reverse-transcribed and amplified using the polymerase chain reaction and the products were cloned and sequenced. Three single base missense mutations were identified: Arg111----Trp, Gln286----Arg, and Arg193----Gln. One single base amber mutation was identified at Gln454. One mutation involved a 13-base pair deletion within exon 13, and it was noted that the majority of the mature RNA derived from this allele was deleted for the entire exon rather than containing the exon with the 13 bases deleted. A final mutation was observed in which exon 2 was deleted from the mature RNA. The molecular basis for this deletion was not determined. Of the eight potential mutations present in the four cell lines studied, six mutations were identified and further data indicate that the remaining two unidentified mutations were different from those identified. Two site-directed mutations were created in the cDNA, Lys51----Asn and His89----Gln, and these were expressed in yeast. The Lys51 mutation caused an approximate 2-fold reduction in activity and the His89 mutation resulted in an approximate 10-fold reduction in activity. The combination of determination of naturally occurring mutations and the study of the effect of site-directed mutations on the activity of argininosuccinate lyase provide insight into the amino acid residues critical to the function of the enzyme.

Amino Acid Sequence↗
PubMed1991

Characterization of the human argininosuccinate lyase gene and analysis of exon skipping.

A cosmid clone containing the entire human argininosuccinate lyase gene was isolated and characterized. The gene is approximately 35 kb in length and is divided into 16 exons. The sequence surrounding all splice sites was determined, and one unusual 5' splice site sequence in which a GC dinucleotide occurred instead of the consensus GT dinucleotide was observed. The exon structure of the gene is identical to that of the rat and similar to that of the delta-crystallin genes in the chicken. The transcription initiation site was identified, and 252 bases 5' of that site were sequenced. Within that region, the GC content is 75%, no TATA element was observed, and multiple potential Spl and CACCC binding sites were noted. Also, a putative consensus sequence that may play a role in the regulation of urea cycle genes was identified within this region. Analysis of the structure of the mature mRNA derived from this gene revealed that the sequences encoded by exon seven were deleted in approximately 5-10% of the mature mRNA in all tissue sources examined.

Argininosuccinate Lyase↗